Staphylococcal toxic shock exotoxin (its isolation and characteristics)

The method for obtaining the preparation of toxic shock exotoxin (TSE) has been developed. This method comprises the following operations: the sorption of the toxin from the culture fluid on Amberlite CG-50, elution, dialysis, gel chromatography in a column with biogel P-2, isoelectric focusing, and gel chromatography in a column with Sephadex G-75. TSE is a relatively thermostable protein with a molecular weight of 24,000. Its isoelectric point is 7.2. Monospecific antiserum to TSE with precipitating antibody titer equal to 1:16, identical to the reference serum (M. S. Bergdoll), has been prepared. This antiserum has shown no cross reactions with the homogeneous preparations of staphylococcal enterotoxins.     

Prion protein glycosylation

The transmissible spongiform encephalopathies (TSE), or prion diseases are a group of transmissible neurodegenerative disorders of humans and animals. Although the infectious agent (the 'prion') has not yet been formally defined at the molecular level, much evidence exists to suggest that the major or sole component is an abnormal isoform of the host encoded prion protein (PrP). Different strains or isolates of the infectious agent exist, which exhibit characteristic disease phenotypes when transmitted to susceptible animals. In the absence of a nucleic acid genome it has been hard to accommodate the existence of TSE strains within the protein-only model of prion replication. Recent work examining the conformation and glycosylation patterns of disease-associated PrP has shown that these post-translational modifications show strain-specific properties and contribute to the molecular basis of TSE strain variation. This article will review the role of glycosylation in the susceptibility of cellular PrP to conversion to the disease-associated conformation and the role of glycosylation as a marker of TSE strain type.     

Molecular basis of scrapie strain glycoform variation

Transmissible spongiform encephalopathies (TSE) are characterized by the conversion of a protease-sensitive host glycoprotein, prion protein or PrP-sen, to a protease-resistant form (PrP-res). PrP-res molecules that accumulate in the brain and lymphoreticular system of the host consist of three differentially glycosylated forms. Analysis of the relative amounts of the PrP-res glycoforms has been used to discriminate TSE strains and has become increasingly important in the differential diagnosis of human TSEs. However, the molecular basis of PrP-res glycoform variation between different TSE agents is unknown. Here we report that PrP-res itself can dictate strain-specific PrP-res glycoforms. The final PrP-res glycoform pattern, however, can be influenced by the cell and significantly altered by subtle changes in the glycosylation state of PrP-sen. Thus, strain-specific PrP-res glycosylation profiles are likely the consequence of a complex interaction between PrP-res, PrP-sen, and the cell and may indicate the cellular compartment in which the strain-specific formation of PrP-res occurs.     

How independent are TSE agents from their hosts?

Central to understanding the nature TSE agents (or prions) is how their genetic information is distinguished from the host. Are TSEs truly infectious diseases with host-independent genomes, or are they aberrations of a host component derived from the host genome? Recent experiments tested whether glycosylation of host PrP affects TSE strain characteristics. Wild-type mice were infected with 3 TSE strains passaged through transgenic mice with PrP devoid of glycans at 1 or both N-glycosylation sites. Strain-specific characteristics of 1 TSE strain changed but did not change for 2 others. Changes resulted from the selection of mutant TSE strains in a novel replicative environment. In general the properties of established TSEs support the genetic independence of TSE agents from the host, and specifically the primary structure of PrP does not directly encode TSE agent properties. However sporadic TSEs, challenge this independency. The prion hypothesis explains emerging TSEs relatively successfully but poorly accounts for the diversity and mutability of established TSE strains, or how many different infectious conformations are sustained thermodynamically. Research on early changes in RNA expression and events at the ribosome may inform the debate on TSE agent properties and their interaction with host cell machinery.     

Host PrP glycosylation: a major factor determining the outcome of prion infection

The expression of the prion protein (PrP) is essential for transmissible spongiform encephalopathy (TSE) or prion diseases to occur, but the underlying mechanism of infection remains unresolved. To address the hypothesis that glycosylation of host PrP is a major factor influencing TSE infection, we have inoculated gene-targeted transgenic mice that have restricted N-linked glycosylation of PrP with three TSE strains. We have uniquely demonstrated that mice expressing only unglycosylated PrP can sustain a TSE infection, despite altered cellular location of the host PrP. Moreover we have shown that brain material from mice infected with TSE that have only unglycosylated PrP^Sc is capable of transmitting infection to wild-type mice, demonstrating that glycosylation of PrP is not essential for establishing infection within a host or for transmitting TSE infectivity to a new host. We have further dissected the requirement of each glycosylation site and have shown that different TSE strains have dramatically different requirements for each of the glycosylation sites of host PrP, and moreover, we have shown that the host PrP has a major role in determining the glycosylation state of de novo generated PrP^Sc.     

Enterotoxigenic E. coli in humans.

E. coli strains isolated from persons having diarrhoeal disease were tested for the production of TS enterotoxin. The production of TSE was demonstrated in 2.5% in a series of 80 strains isolated from children under one year of age, having acute diarrhoea. TSE was produced by 8.4% of E. coli strains out of 59 strains isolated from patients over one year of age. Among these strains, an interesting E. coli strain was isolated from the patient T. J., which produced TSE for more than 15 months. The production of TLE was tested though not proved in all strains by experiment on an isolated intestinal loop of an adult rabbit. The test on suckling mice so far appears to be the most suitable test for the demonstration of TSE. The results were considered positive when the index (the ratio of the weight of the whole intestine to the weight of the rest of the body) was higher than 0.08 while indices up to 0.078 were considered negative. E. coli strains with indices of intermediate values and strains with temporary production of TSE, occurring particularly in very small children, deserve special attention. The height of the indices was not influenced by a 30-minute exposure at 60 degrees C, but a decrease in the values of the indices was observed after boiling for a period of 15 min. The occurrence of E. coli strains producing TSE is evidently small in humans in European countries but, without doubt, they are important in the aetiology of diarrhoeal diseases.     

Single radial immunodiffusion method for screening Staphylococcal isolates for enterotoxin.

A direct system for screening large numbers of staphylococcal isolates for enterotoxin production has been developed. The system employs polyvalent (serotypes A, B, C, D, and E) immunodiffusion assay slides in conjunction with a multiple-culturing system for toxin production. With the combined system, as many as 50 cultures can be screened simultaneously on a single assay slide having a sensitivity of about 0.3 microgram/ml. The system should be useful for detecting potential enterotoxin in foods containing a predominance of non-enterotoxigenic strains.     

Antiseptic resistance of clinical strains of Staphylococcus aureus

To evaluate the activity of antiseptics and the sensitivity-resistance of bacteria to antiseptics, a number of characteristics has been used, including the minimum inhibiting concentration for different strains, the frequency of statistical and clinical resistance, the antiseptic activity index. A wide spread of S. aureus strains isolated from patients with hospital infections has been revealed. Differences in the resistance of bacterial strains have been established, depending on the type of the antiseptic and the ecovar of bacteria: among hospital ecovars, resistant strains occur more frequently and can resist a wider range of antibiotics. In staphylococcal hospital ecovars the occurrence and level of resistance to a number of widely used antiseptics increase with time. In connection with a wide spread of staphylococcal hospital strains resistant to antiseptics, measures on the control of the circulation of such strains should be introduced into hospitals, and the data thus obtained should be used for the periodic reevaluation of antiseptics used in medical practice and for the choice of preparations to be used for individual therapeutic and prophylactic antisepsis.     

Characterization of thermodynamic diversity between transmissible spongiform encephalopathy agent strains and its theoretical implications.

Some transmissible spongiform encephalopathy (TSE) (or "prion") strains, notably those derived from bovine spongiform encephalopathy, are highly resistant to total inactivation by heat. When three TSE strains derived from sheep with scrapie were heated, little inactivation took place at low temperatures, but at higher temperatures, considerable inactivation occurred. The temperature at which substantial inactivation first occurred varied according to TSE strain, and it was calculated to be 70 degrees C for the 22C strain, 84 degrees C for ME7, and 97 degrees C for 22A by fitting the data to a model based on competition between a destructive and a protective reaction. However, PrP(Sc) from mice infected with a range of TSE strains retained similar resistance to proteinase K digestion after heating to below or above these temperatures, showing that the properties of PrP(Sc) responsible for proteinase resistance do not correlate with those conferring thermostability on the TSE agent. The simplest explanation of these data is that the causal agent contains a macromolecular component that is structurally independent of the host, that it varies covalently between TSE strains, and that it is protected by other macromolecular components. The model is in accord with the virino hypothesis, which proposes a host-independent informational molecule protected by the host protein PrP.     

Post‐translational changes to PrP alter transmissible spongiform encephalopathy strain properties

The agents responsible for transmissible spongiform encephalopathies (TSEs), or prion diseases, contain as a major component PrP^Sc, an abnormal conformer of the host glycoprotein PrP^C. TSE agents are distinguished by differences in phenotypic properties in the host, which nevertheless can contain PrP^Sc with the same amino‐acid sequence. If PrP alone carries information defining strain properties, these must be encoded by post‐translational events. Here we investigated whether the glycosylation status of host PrP affects TSE strain characteristics. We inoculated wild‐type mice with three TSE strains passaged through transgenic mice with PrP devoid of glycans at the first, second or both N‐glycosylation sites. We compared the infectious properties of the emerging isolates with TSE strains passaged in wild‐type mice by in vivo strain typing and by the standard scrapie cell assay in vitro. Strain‐specific characteristics of the 79A TSE strain changed when PrP^Sc was devoid of one or both glycans. Thus infectious properties of a TSE strain can be altered by post‐translational changes to PrP which we propose result in the selection of mutant TSE strains.     

Formation of protease-resistant prion protein in cell-free systems

In transmissible spongiform encephalopathies (TSE) or prion diseases, the endogenous protease-sensitive prion protein (PrP-sen) of the host is converted to an abnormal pathogenic form that has a characteristic partial protease resistance (PrP-res). Studies with cell-free reactions indicate that the PrP-res itself can directly induce this conversion of PrP-sen. This PrP-res induced conversion reaction is highly specific in ways that might account at the molecular level for TSE species barriers, polymorphism barriers, and strains. Not only has this reaction been observed using mostly purified PrP-sen and PrP-res reactants, but also in TSE-infected brain slices. The conversion mechanism appears to involve both the binding of PrP-sen to polymeric PrP-res and a conformational change that results in incorporation into the PrP-res polymer.     

Inhibition of Leukocyte Migration by a Staphylococcal Factor

Cell wall mucopeptide isolated from virulent strains of Staphylococcus aureus has previously been found to potentiate subcutaneous staphylococcal lesions in mice. This cell wall fraction was found to inhibit the migration of polymorphonuclear leukocytes toward a chemotactic stimulus, as tested by the micropore filter chamber technique. A close correlation was shown to exist between in vivo "mouse virulence" of staphylococcal strains and the in vitro inhibition of leukocyte migration by the cell wall factor.     

Variability of the pathogenicity traits of populations of staphylococci in a surgical hospital

The results of the study of heterogeneity of staphylococcal populations at a surgical ward are presented. The study deals with qualitative and quantitative characteristics of three groups of pathogenicity factors: protease (the penetration factor), protein A (the function of protection from phagocytosis) and alpha-hemolysin (the toxic function). The study shows that the greatest number of S. aureus strains with a high content of protein A has been isolated from patients with postoperative and wound infections. On the basis of the data obtained in this study the groups of strains have been defined in accordance with the association of the signs of pathogenicity. These groups reflect pronounced heterogeneity of staphylococcal strains at a surgical ward.     

Staphylococcal adherence to rabbit epithelial cells

The adherence of Staphylococcus aureus strains to rabbit epithelial cells has been studied. The strains have been shown to possess similar adhesiveness with respect to the epithelium of the mouth cavity of rabbits. The investigation, carried out with the use of one staphylococcal strain taken as a model, has revealed that the cells of this strain adhere to different areas of the epithelium in the mouth cavity in varying amounts, the amount of adhering bacteria depending on the age of rabbits. The data presented in this work suggest that in staphylococci adhering to rabbit epithelial cells the adhesive function is performed by thermostable and trypsin-resistant staphylococcal cell-wall surface structures of nonprotein nature.     

The functional interchangeability of enterobacterial and staphylococcal iron chelators

The functional interchangeability of staphylococcal and enterobacterial iron chelators was investigated with an indicator system in which minimally effective concentrations of ethylene diamine di-ortho-hydroxyphenyl acetic acid (EDDA) were used to inhibit the growth of indicator strains in the depth of simple agar media by making the iron unavailable. Test colonies were then applied to the surface of the media to determine whether the indicator organisms, by utilising chelators from the test colony could obtain the required iron for growth, in its vicinity.Approximately 50% of staphylococcal strains, both S. aureus and S. epidermidis, reversed the inhibition of enterobacterial indicators, whereas almost all enterobacterial test strains, representing five genera, reversed the inhibition of the staphylococcal indicators. A purified preparation of the enterobacterial iron chelator enterochelin also reversed the inhibition of four out of the five staphylococcal indicator strains.     

Expression of δ-toxin by Staphylococcus aureus mediates escape from phago-endosomes of human epithelial and endothelial cells in the presence of β-toxin

Staphylococcus aureus is able to invade non-professional phagocytes by interaction of staphylococcal adhesins with extracellular proteins of mammalian cells and eventually resides in acidified phago-endosomes. Some staphylococcal strains have been shown to subsequently escape from this compartment. A functional agr quorum-sensing system is needed for phagosomal escape. However, the nature of this agr dependency as well as the toxins involved in disruption of the phagosomal membrane are unknown. Using a novel technique to detect vesicular escape of S. aureus, we identified staphylococcal virulence factors involved in phagosomal escape. Here we show that a synergistic activity of the cytolytic peptide, staphylococcal δ-toxin and the sphingomyelinase β-toxin enable the phagosomal escape of staphylococci in human epithelial as well as in endothelial cells. The agr dependency of this process can be directly explained by the location of the structural gene for δ-toxin within the agr effector RNAIII.     

Membrane protein spectra of staphylococci from a comparative physiological aspect]

Membrane protein spectra, obtained by electrophoresis in the system polyacrylamide gel--sodium dodecysolfate, were studied in 14 staphylococcal strains with different properties. The proteinograms of membranes were shown to be useful as an additional criterion in the identification of staphylococci. Fractions and their combination characteristic of all the strains and their separate groups were revealed in the protein spectra of staphylococcal membranes.